WO2007009404A1 - Rh-epo nasal formulations with low sialic acid concentration for the treatment of diseases of the central nervous system - Google Patents

Abstract

The invention relates to the biopharmaceutical industry and, in particular, to the development of a medicament which is intended for the treatment of cerebrovascular, neurodegenerative and psychiatric diseases and which contains, by way of active principle, recombinant human erythropoietin (rh-Epo) with a low sialic acid concentration. During rh-Epo production, the glycoprotein is obtained with a different sialic acid concentration when less than 40 % of the molecule thereof is protected with (basic) sialic acid, in which case it is not biologically active by systemic route and is inactivated by hepatic enzymes. Surprisingly, it was found that nasally-administered basic rh-Epo has greater therapeutic benefits than the acid. The inventive basic rh-Epo nasal formulations include bioadhesive polymers which increase the residence time in the nasal cavity, thereby enhancing the therapeutic effect thereof. Said formulations also include other auxiliary substances, such as preservative substances, surfactants, pH regulators, isotonising agents and protein stabilisers.

Description

NASAL FORMULATIONS OF EPOrh WITH LOW SYNALIC ACID CONTENT FOR THE TREATMENT OF CENTRAL NERVOUS SYSTEM DISEASES.

Technical Sector

The present invention relates to the Biopharmaceutical Industry and in particular to the production of a medicament for nasal administration of EPOrh with low content of sialic acid (basic) from Chinese hamster ovary cells by genetic engineering for the treatment of cerebrovascular, neurodegenerative and psychiatric diseases. Previous Technique

Erythropoietin (EPO) is a glycoprotein hormone whose molecular weight ranges around 34kDa; The protein fraction consists of approximately 166 amino acids; It occurs in the kidney, liver and cells of the central nervous system and is involved in the proliferation, differentiation and maturation of erythrocytes and other hematopoietic cells.

In 1998, Sakanaka and collaborators reported the neuroprotective properties of EPOrh against ischemic damage in vivo, after occlusion of the common carotid artery, a model of global ischemia followed by infusion of EPOrh in the lateral ventricles of gerbils, the authors observed a reduction of ischemic damage on CA1 hippocampal neurons.

The neuroprotective effects of EPOrh may be due to different factors within which they are: antagonism of glutamate-induced cytotoxicity, increase in the expression of antioxidant enzymes, decrease in the formation of free radical-mediated nitric oxide (NO), normalization of cerebral blood flow, influence on the release of neurotransmitters, promotion of neoangiogenesis, inhibition of apoptosis induced by excitotoxins and NO, increase in the expression of antiapoptotic genes, neurotropic effect, decrease in neuronal excitability, cerebral anti-inflammatory effect, inhibition of The apoptosis induced by kainate and the production of proinflammatory atokines, angiogenic and neurogenic effect and contributes to ischemic preconditioning and as a whole can justify the use of EPOrh in the treatment of cerebrovascular, neurodegenerative and psychiatric diseases.

The neuroprotective properties of EPOrh can prevent or reverse the neuronal death characteristic of many diseases of the central nervous system, such as ischemia, neurotrauma, and neurodegenerative and neuroinflammatory diseases.

It has also been described that neurodegenerative processes contribute to the triggering of the pathophysiology of schizophrenia, which is why neuroprotective drugs can be an effective therapeutic alternative for the treatment of this disease.

Once the organism is exposed to different pathological states such as hypoglycemia, strong neuronal depolarization or by the generation of reactive oxygen species by mitochondria, the expression of EPO increases significantly.

On the other hand, inflammation increases brain injury by several mechanisms, even directly inhibiting the production of local EPO. In fact, although EPO is endogenously produced in the brain after ischemia, its expression is markedly inhibited by inflammatory cytokines. Said inhibition decreases the neuroprotective capacity of EPO and justifies why the exogenous administration of EPOrh can be especially beneficial for the treatment of cerebrovascular, neurodegenerative and psychiatric disorders.

On the other hand, it is known that to the extent that the EPO molecule has less percent sialic acid, the greater its affinity with its receptor, thus facilitating the physiological response.

The neuroprotective effect of the EPOrh applied intravenously in murine models of cerebral ischemia [LM Brines and cois, PNAS97 (19): 10526-10531, 2000], reduces the damage due to ischemia between 50 and 75%, as well as its application in humans (WO 0354475). However, the clinical and experimental results that use recombinant human acid EPOrh have not assessed the negative impact that Ia may have. medium-term use of this molecule on the induction of erythropoiesis, since, given the low permeability of the blood-brain barrier (BHE) and the low affinity of EPO receptors in the CNS, relatively large amounts of EPOrh are required for a relatively long time to achieve the pharmacological effect. Under these conditions, there is a risk that the viscosity of the blood will be increased and with it the chances of thrombolytic events that trigger cerebral ischemia. These negative consequences of the application of acid EPO have been reported as complications in nephritic patients treated with this molecule. Studies have been developed aimed at obtaining methods that increase the permeability of the blood brain barrier, which is considered a critical step in the incorporation of substances into brain tissue (US 5260308; WO 8901343; US 4801575; US 5442043; JP 57146710 and JP 01149718). In many of these methods the so-called carrier or transporters (US 5442043) are involved forming a peptide conjugate, or they use chimeric peptides (US 48015575) for the incorporation into the brain of the substances. The liposome preparation containing erythropoietin has been reported (JP 08231417).

The use of the nasal route to deliver molecules to the CNS has been reported for gangliosides (US 4639437), as well as for neuronal therapeutic agents that reach the brain through absorption by the nasal mucosa and its subsequent transport through the Neural olfactory pathway (EP 504263).

There are several patents related to the protection of liquid formulations of EPOrh, the following are the most representative: Liquid preparations where the EPOrh is used as an active ingredient, and the protein in all cases is presented as an injectable (example: CIPO- 2041989; CIPO-2353553), this route of administration is indicative that the EPO used is acidic, that is, it must have more than 40% of the molecule covered with sialic acid, because otherwise it is inactivated by the liver enzymes and It would not be clinically effective.

The CIPO-2074820 patent uses the nasal route for the administration of the protein, in this case cyclodextrin is used as a component of the formulation and also pursues a systemic absorption of EPO, so the protein also has to be acidic.

There are scientific publications that refer to the use of biodhesives to increase the residence time of proteins in the nasal cavity [Polireddy D et cois, International Journal of Pharmaceutics (127): 115-133, 1996], however, there are no reports of their use in formulations containing EPOrh.

The CIPO-2353553 patent justifies the exogenous administration of the EPO for the treatment of cerebral ischemia, but as in the previous cases it is pursued that the protein passes directly or indirectly to the bloodstream to be effective, so that we are also in presence of EPO with high sialic acid content.

The CIPO-2408685 patent protects different EPO formulations, but its use is intended for the treatment of anemia, which is why it is acidic EPO, since the basic EPO does not promote the proliferation, differentiation and maturation of erythrocytes.

The CIPO-2437333 patent specifies that the protected formulations are from epoetin alfa, that is to say acid EPO.

In all the cases described above and reviewed in the literature, reference is made to the acid EPOrh, our patent pursues the protection of liquid formulations from basic EPOrh. Disclosure of the Invention

During the production of EPOrh, the glycoprotein with different sialic acid content is obtained, when the protein has less than 40% of its molecule protected with sialic acid (basic), it is eliminated because it is not biologically active systemically. This basic or low sialic acid EPOrh represents 70% of the total EPOrh production. Surprisingly we have found that the basic EPOrh administered by the nasal route may have therapeutic utilities superior to the acid; The results of this invention allow us to take advantage of the basic isoforms of the EPOrh that are currently not recovered and are easily acquired. This represents a great economic advantage because it allows recovering what is currently a waste of the production of EPOrh.

This formulation can be used for the treatment and / or prevention of diseases of the central nervous system including cerebrovascular, neurodegenerative and psychiatric disorders, on an outpatient basis at the level of primary health care. The advantages of the proposed solution include the use of EPOrh with low sialic acid content for therapeutic purposes allowing a greater protective effect with lower doses. A rapid arrival at the site of action is achieved, which is critical when it comes to achieving a therapeutic effect against cerebral hypoxia. In the specific case of cerebrovascular diseases, in the acute phase it is necessary to start the therapeutic intervention before certain processes of the ischemic cascade have occurred, which is called the therapeutic window period.

The nasal route of administration of basic EPOrh eliminates the risk of inducing erythropoiesis. An increase in the concentration of erythrocytes increases the viscosity of the blood and compromises reperfusion after an occlusive or hemorrhagic episode, decreases cerebral blood flow and therefore hinders the arrival of oxygen and nutrients to the tissue.

The nasal route has greater advantages than other forms of administration, allowing rapid access to the brain, it is less invasive than the intravenous or intracerebroventricular one and a percentage of the EPOrh can reach the cerebrospinal fluid without having to pass the bloodstream before avoiding the hepatic first-pass metabolism and consequently its inactivation. The nasal application route is not traumatic and eliminates surgical risks or other possible implications given by traumatic routes. It constitutes an alternative route of access to the brain without damaging the blood brain barrier. The use of an alternative route to the vascular route ensures the arrival of the molecule to poor or non-irrigated areas of the central nervous system, by diffusion through the interstitial fluid.

Taking into account that one of the main limitations of the use of the nasal route is the rapid mucociliary clearance, the use of bioadhesive polymers that increase the residence time of the EPOrh in Ia is followed as a strategy in this invention. Nasal Cavity. During the development of the formulation, other auxiliary substances or excipients are also incorporated, such as preservative substances, surfactants, pH regulators, isotonic agents and protein stabilizers; to obtain a stable physicochemical and microbiological formulation that maintains its therapeutic properties over time.

An EPOrh was used that contained in its molecule less than 40% sialic acid (basic) at a concentration from 0.5 to 2 mg / mL. The formulations presented are liquid, colorless, transparent and free of mechanical impurities preparations with an apparent viscosity that ranges from 10 to 250 mPas using hydroxypropylmethylcellulose (HPMC) as bioadhesive polymers in concentrations from 0.4 to 0.9%, hydroxypropylcellulose , (HPC) 0.2 to 0.8% and methylcellulose (MC) from 0.25 to 0.5%.

The pH was adjusted in a range from 6.0 to 7.5 using mainly phosphate buffers and citrates in concentrations of 20 to 100 mM / L.

Osmotic pressure can be regulated with different isotonizers among which are: sodium chloride, mannitol, sorbitol, glucose, among others, in a range of 0.05 to 10 g / L

The preservative agents were: benzalkonium chloride of 0.01 to 0.02% in combination with disodium EDTA in concentrations of 0.01%, chlorobutanol of 0.3 to 0.5%, methylparaben of 0.02 to 0.035% and propylparaben of 0.01 to 0.02%.

To avoid the adhesion of the protein to the walls of the packaging material, nonionic surface active substances such as polyethylene sorbitan laurate (tween 20 to 80), cremofor RH 40, sorbitan trioleate (spam 35 to 80) were used, in a range of concentrations between 0.01 and 1 g / L.

Different amino acids were used as protein stabilizers, among which are: L-tryptophan, L-leucine, L-arginine hydrochloride and L- hydrochloride histidine in a concentration range between 0.1 and 10 mg / mL The solvent used in all cases was water for injection.

Irritability tests of the nasal mucosa were performed in rats, with no evidence of irritation at this level, which, added with the magnificent tolerance of the product, supports the use of the basic EPOrh in humans from the toxicological point of view.

Stability studies showed an adequate behavior of protein concentrations over time after using the lowry method (± 10%), the pH was kept within the established limit.

EXAMPLES OF EMBODIMENT Example 1:

Purification of the EPOrh with low sialic acid content:

To obtain the basic erythropoietin with sialic acid content less than 40%, three chromatographic steps were basically carried out, based on a material of great complexity and with a large amount of contaminating proteins, since it contains 5% of fetal bovine serum. Subsequently, an additional step is performed to obtain the isoforms of interest.

For the diafiltration and concentration of the basic EPOrh, a laboratory scale ultrafiltration system (SARTOFLOW SLICE 200) was used, coupled to a peristaltic pump (IP55 Watson-Marlow). A 10 kDa Hydrosart membrane was used. A flow of 1.9 ± 0.25 imL / min, a feed pressure (P1) of 2.9 ± 0.25 bar, a hold pressure (P2) of 0.65 ± 0.1 bar and a transmembrane pressure (TM) of 1.8 ± 0.2 bar were worked . To determine the protein concentration, a Spectrophotometer (Ultrospec ™ 3100) was used at λ = 280 nm. For the diafiltration process, a nasal buffer solution of NaH ₂ PO ₄ and Na ₂ HPO _{4 was used} at a pH of 6.0 and a conductivity of 3.25 ms / cm. The pH was controlled using a Sartorius pH meter and an Omega conductimeter. The process performance is in a range between 75 - 95%. Table 1 shows the results obtained in the process:

Example 2:

EPOrh nasal formulation with low sialic acid content that uses HPMC bioadhesive polymer, histidine hydrochloride as a stabilizer, Tween 80 as a surface active substance and benzalkonium chloride as a preservative.

Preparation method:

For the preparation of the solution, it is based on a volume of water for injection that represents 30% of the total volume of the formulation, it preserves the buffer, the buffers and the isotonizing agent. Moreover in a container of suitable capacity 15% of the total volume of the formulation of water for injection they are incorporated and heated 85-95 ⁰ C, pulverizing the polymer with stirring strong and constant. Once the polymer has been wetted, the previously prepared solution is incorporated thereto with vigorous stirring until total homogenization. The agitation is reduced for the incorporation of the corresponding volume of basic EPOrh, the superficially active substance and the protein stabilizer, finally it is flush with water for injection to a final volume that represents 100% of the formulation. The formulation is filtered through a 0.2 μm cellulose acetate membrane and it is verified that the pH is maintained in the established range. The composition of the finished form is as follows:

Proportion Component

Basic EPOrh 0.8 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 80 0.25 mg / mL

Sodium Chloride 7.4 mg / mL Ethylenediamine Tetraacetic Acid 0.1 mg / mL Disodium

Benzalkonium Chloride 0.2 mg / mL

Water for injection csp 1 mi

Example 3:

Nasal formulation of EPOrh with a low sialic acid content similar to the previous example but with methylcellulose as a bioadhesive polymer Preparation method (similar to example 2):

The composition of the finished form is as follows:

Component Proportion

Basic EPOrh 0.8 mg / mL

Methylcellulose 3.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 80 0.25 mg / mL

Sodium Chloride 7.4 mg / mL Ethylenediamine Tetraacetic Acid 0.1 mg / mL Disodium

Benzalkonium Chloride 0.2 mg / mL

Water for injection csp 1 mi Example 4:

EPOrh nasal formulation with low sialic acid content similar to example 2 but with Tween 20 as a superficially active substance.

Preparation method (similar to example 2):

The composition of the finished form is as follows:

Proportion Component

Basic EPOrh 0.8 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 20 0.20 mg / mL

Sodium Chloride 7.4 mg / mL Ethylenediamine Tetraacetic Acid 0.1 mg / mL Disodium

Benzalkonium Chloride 0.2 mg / mL

Water for injection. csp 1 mi

Example 5:

EPOrh nasal formulation with low sialic acid content similar to the previous example but with 1.2 mg / ml of active substance and without histidine hydrochloride as a protein stabilizer. Preparation method (similar to example 2):

The composition of the finished form is as follows:

Component Proportion

Basic EPOrh 1.2 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 20 0.20 mg / mL

Sodium Chloride 7.7 mg / mL Ethylenediamine tetraacetic acid 0.1 mg / mL disodium

Benzalkonium Chloride 0.2 mg / mL

Water for injection csp 1 mi

Example 6:

EPOrh nasal formulation with low sialic acid content similar to example 2 but with 1.2 mg / ml of active substance and without histidine hydrochloride as a protein stabilizer.

Preparation method (similar to example 2, without incorporating histidine hydrochloride):

The composition of the finished form is as follows:

Component Proportion

Basic EPOrh 1.2 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 80 0.25 mg / mL

Sodium Chloride 7.7 mg / mL

Ethylenediamine tetraacetic acid 0.1 mg / mL disodium

Benzalkonium Chloride 0.2 mg / mL

Water for injection csp 1 mi

Example 7:

Formulation from EPOrh with low content of sialic acid in the form of nasal administration similar to example 2 but with methylparaben and propylparaben as antimicrobial preservatives.

Preparation method:

For the preparation of the solution, it is based on a volume of water for injection that represents 30% of the total volume of the formulation, in it the buffers are solubilized and the agent toner. Moreover in a container of suitable capacity 15% of the total volume of the formulation of water for injection are incorporated and heated between 90-95 ⁰ C, the parabens are dissolved and the polymer incorporated by stirring strong and constant. Once the polymer has been wetted, the previously prepared solution is added thereto with vigorous stirring until total homogenization. The agitation is reduced for the incorporation of the corresponding volume of basic EPOrh, the superficially active substance and histidine hydrochloride and finally it is flush with water for injection to a final volume that represents 100% of the formulation. Subsequently, the formulation is filtered through a 0.2μm cellulose acetate membrane and it is verified that the pH is maintained in the established range.

The composition of the finished form is as follows:

Component Proportion

Basic EPOrh 0.8 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 80 0.25 mg / mL

Sodium Chloride 7.5 mg / mL

Methylparaben 0.33 mg / mL

Propylparaben 0.17 mg / mL

Water for injection csp 1 mL

Example 8:

EPOrh nasal formulation with low sialic acid content similar to the previous example but with methylcellulose as bioadhesive polymer.

Preparation method (similar to example 6): The composition of the finished form is as follows:

Component Proportion

Basic EPOrh 0.8 mg / mL

Methylcellulose 3.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 80 0.25 mg / mL

Sodium Chloride 7.5 mg / mL

Methylparaben 0.33 mg / mL

Propylparaben 0.17 mg / mL

Water for injection csp 1 mL

Example 9:

Nasal formulation of EPOrh with low sialic acid content similar to example 7 but with Tween 20 as a surface active substance Preparation method (similar to example 7):

The composition of the finished form is as follows:

Component Proportion

Basic EPOrh 0.8 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 20 0.20 mg / mL

Sodium Chloride 7.5 mg / mL

Methylparaben 0.33 mg / mL

Propylparaben 0.17 mg / mL

Water for injection csp 1 mL Example 10:

Formulation from EPOrh with low sialic acid content similar to the previous example but without histidine hydrochloride as stabilizing agent and glucose as isotonic.

Preparation method (similar to example 7):

The composition of the finished form is as follows:

Proportion Component

Basic EPOrh 1.2 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 20 0.20 mg / mL

Glucose 48.0 mg / mL

Methylparaben 0.33 mg / mL

Propylparaben 0.17 mg / mL

Water for injection csp 1 mL

Example 11:

Formulation from EPOrh with low sialic acid content similar to the previous example but with Tween 80 as a surface active substance.

Preparation method (similar to example 7):

The composition of the finished form is as follows:

Proportion Component

Basic EPOrh 1.2 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 80 0.25 mg / mL

Glucose 48.0 mg / mL Methylparaben 0.33 mg / mL

Propylparaben 0.17 mg / mL

Water for injection csp 1 ml_

Example 12: Nasal formulation of EPOrh with low sialic acid content similar to example 7 but with chlorobutanol as an antimicrobial preservative.

Preparation method (similar to example 2):

The composition of the finished form is as follows:

Proportion Component

Basic EPOrh 0.8 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 80 0.25 mg / mL

Sodium Chloride 6.2 mg / mL

Chlorobutanol 5.0 mg / mL

Water for injection csp 1 mL

Example 13:

Formulation from EPOrh with low sialic acid content similar to the previous example but with methylcellulose as bioadhesive polymer.

Preparation method (similar to example 2): The composition of the finished form is as follows:

Proportion Component

Basic EPOrh 0.8 mg / mL

Methylcellulose 3.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 80 0.25 mg / mL

Sodium Chloride 6.2 mg / mL

Chlorobutanol 5.0 mg / mL

Water for injection csp 1 mL

Example 14:

EPOrh nasal formulation with low sialic acid content similar to example 12 but with Tween 20 as a superficially active substance.

Preparation method (similar to example 2):

The composition of the finished form is as follows:

Proportion Component

Basic EPOrh 0.8 mg / mL

Hydroxypropyl methylcellulose 5.0 mg / mL

Histidine Hydrochloride 1.0 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 20 0.20 mg / mL

Sodium Chloride 6.2 mg / mL

Chlorobutanol 5.0 mg / mL

Water for injection csp 1 mL Example 15:

EPOrh nasal formulation with low sialic acid content similar to example 5 but with chlorobutanol as an antimicrobial preservative.

Preparation method (similar to example 2):

The composition of the finished form is as follows:

Proportion Component

Basic EPOrh 1.2 mg / mL

Hydroxypropyl methylcellulose 5 mg / mL

Sodium Dihydrogen Phosphate 2 mg / mL

Disodium hydrogen phosphate 0.7 mg / mL

Tween 20 0.20 mg / mL

Sodium Chloride 6.5 mg / mL

Chlorobutanol 5.0 mg / mL

Water for injection csp 1 mL

Example 16: "In vitro" evaluation of the stimulatory action on cells of nervous origin of the nasal formulation of recombinant human erythropoietin with a low content of sialic acid

To demonstrate the inductive effect on the specific activity of the Glutathione-S-Transferase (GST) enzyme of EPO in cells of nervous origin, as well as to demonstrate that recombinant human acid and basic Erythropoietin have a similar effect on the cells of nervous origin, an in vitro study was performed using PC12 cells. The cells were maintained undifferentiated in DMEM medium supplemented with 2 mM L-Glutamine, 100 Units / ml penicillin G, 100 micrograms / ml streptomycin (GIBCO), 10% (v / v) containing 1OmM Hepes Buffer. In the culture 10% total horse serum was used. To the cells were added 0.6 and 15 nanomolar recombinant human Erythropoietin with low sialic acid content (examples 5 and 6) and / or acid erythropoietin. As controls, similar amounts of EPO previously heat inactivated were added. The values of the specific activity of the GST obtained in these cultures was considered as 100%. For the determination of the specific activity of the GST, the homogenate of the cells grown with acidic or basic EPO was used, which was performed in an all-glass homogenization system at 4 degrees celcius, in 0.1 M phosphate buffer pH 6 , 95. The cell-free homogenate was obtained by centrifugation at 16,000 X at 4 degrees celcius in a high speed refrigerated centrifuge. The homogenate thus obtained was kept in an ice bath and was used for the determination of the specific activity of the GST according to the Habig method. (Habig, WH, Pabst, JJ, and Jakoby, WB 1974; Biol. Chem. 249 (22): 7130-7139). Protein determination was performed by the Bradford method (Bradford MM. Anal. Biochem, 72, (176) 158). As a main result, it was obtained that the cells of nervous origin responded to the presence of the EPO by modifying the activity of the GST enzyme. The plotted values (Fig. 1) represent the average value of three independent crops. This graph shows the action of acid and basic erythropoietin on the enzymatic activity of GST in PC12 cell culture. In this graph we can observe how the cells of nervous origin respond to the presence of the EPO by modifying the activity of the GST enzyme. The response with recombinant human Erythropoietin with low sialic acid and acid content was similar.

Example 17:

Therapeutic efficacy of EPOrh with low sialic acid content administered by the nasal route.

Mongolian gerbils of both sexes were used, with a body weight between 60 and 70 grams, with water and ad limitum feeding, which were maintained in a 12-hour alternating cycle of light-dark throughout the experimental period.

The unilateral permanent occlusion (ULP) of the right carotid of the animals was performed, under deep anesthesia, for which the intraperitoneal route was used, as a diazepan pre-anesthetic (5.0 mg / Kg.); as a ketamine anesthetic (47 mg / kg) and 0.02 mg / kg of Atropine. The right carotid was exposed under stereoscope, doubly linked and sectioned. The falsely injured animals were obtained with the same manipulations but without binding or cutting the carotid. For the clinical and histopathological evaluation, the experimental groups were: Control Group: isolated carotid, with no other procedure (false injured). (n = 10) Group of Injured Animals without treatment: Injured animals, without other procedure (n = 20).

Group of Injured Animals with treatment: isolated carotid, doubly linked and sectioned, with administration of EPOrh with low content of sialic acid by nasal route (example 10) (n = 20).

The treatment consisted of the application in the nasal cavity of 10 μl of EPOrh or its vehicle every 8 h daily, from 1 hour after the operation until the 4th. day after the operation.

To each animal the neurological state was determined by a scale of 2 to 5 that included body tone, prehensile strength and alterations in posture and gait. A surviving animal without pathological signs has a value of 5 on this scale.

For the functional evaluation the spontaneous exploratory activity was also used, where the steepness on the hind legs that the rodents make when exploring a new vessel were counted. A vertical cylindrical box 30 cm in diameter and 25 cm high was used as a container. Each gerbil was placed in the center of that box and the steepings were counted in an interval of 3 minutes.

At 7 days after the operation, the animals were perfused through the left ventricle with 4% formaldehyde in phosphate buffer solution (PBS) at pH 7.0. Brains were extracted and maintained in this solution for a few days. They were subsequently included in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin. The sections were evaluated at 10x and 4Ox, without prior knowledge of their identity.

For the evaluation of cerebral edema, 90 females between 60 and 70 g included in the 3 experimental groups were used: False injured (n = 20) Injured without treatment (n = 35) Injured with treatment (n = 35)

At 3, 12 and 24 hours after the injury the animals were anesthetized and perfused with saline solution, the brain was extracted from the cranial cavity and the hemispheres were separated. The water content was determined according to the gravimetric method described by Calapai and cois, 2000 according to the equation:% Water = [(Weight

Wet of the Hemisphere - Dry weight of the Hemisphere) X 100 X Wet Weight ^"1 ]. For the analysis of the data, differences between groups and samples were established for each variable by the Student t test, ANOVA (one way) and the Dunnett for Ia

Multiple Comparison

To evaluate the body weight of the animals, males were used that were located in 3 groups (of 10 animals each) following the same treatment scheme used in the previous experiments. The animals were weighed on a Sartorius scale on the day of the operation (ULP) and for 5 consecutive weeks (once a week).

During the experiments described where EPOrh was administered by nasal route, a lower mortality was found in the animals treated during the days after the surgery in both sexes, evidenced in the analysis of the proportions. In Figure 2, we can observe the effect on Mortality in both sexes in the model of cerebral ischemia in the gerbil of Mongolia.

At 24 h of the unilateral ligation, a part of the animals showed affectations in the neurological state that were revealed by the examination described and expressed in the score. In Figure 3 we can see the motor behavior of the gerbil of Mongolia in the cylinder. Significant functional damage appears in injured animals without treatment. Exploratory-motor activity appears depressed in untreated ischemic animals while in those treated with EPOrh, it remains similar to that of controls. Figure 4 shows the neurological state of the animals 24 hours after the injury. The effect of the lesion and treatment with EPOrh by nasal route in the model of permanent unilateral ischemia on the body weight of the animals after 5 weeks is shown in Figure 5. The group of injured animals compared with the injured and treated groups or falsely injured showed a different weight curve behavior. Similar curves were obtained in the control and injured group treated, contrasting with the weight loss of the injured and untreated animals. This group could not exceed the average weight with which the experiment began.

The results obtained in the reduction of cerebral edema were due to the fact that the application of EPOrh by nasal route produced a significant protection on the formation of cerebral edema in all treated animals (Fig. 6a and 6b) without finding differences in water content between the animal treated with EPOrh and the controls. A diametrically opposite situation was detected in the group of untreated injured animals where the water content in the injured hemisphere (right) increased at all times studied (3, 12 and 24 hours), finding a high significance at 24 h of the lesion (p <0.001).

In Figure 7 we can observe the histopathological score of the lesions in CA1 of the gerbil hippocampus. In histopathological findings, the frequency of animals with the intact brain is higher in control animals than in animals without treatment (p = 0.002) and is higher in animals treated with EPO than in untreated animals (p = 0.03).

Figure 8 shows M icrofotog raffias of the Gerbil hippocampus treated with EPOrh by nasal route. The integrity of the CA1 sector in the treated animals can be noted. In the untreated animals a generalized edema, pichnosis was observed throughout the right hemisphere as well as hemorrhages in the fimbria of the right hippocampus and in the parietal cortex of the right hemisphere, areas of edema, dense and diffuse chromatin in the right hemisphere. Pycnotic neurons in the hippocampus. Example 18:

"In vitro" evaluation of the antioxidant capacity of erythropoietin with a low content of basic recombinant human sialic acid (example 10) in homogenate of anatomical regions of the hippocampus, cerebral cortex and cerebellum of the rat.

To demonstrate that the acidic and basic EPO are capable of counteracting the production of Malondialdehyde (MDA) and 4-Hydroxyalkenal (4HDA), in rat brain homogenate, an in vivo experiment was performed. For this, male Wistar rats (n = 15) were used, with a body weight between 180 and 200 grams. Water and food were ad libitum, a 12-hour cycle of dark light was given to the animals throughout the experimental period. To obtain the anatomical regions, the following methodology was followed:

The animals were sacrificed by cervical dislocation and decapitation, the brain was quickly removed from the cranial cavity and the tissue was immediately frozen at -70 degrees celcius. The hippocampus, cerebral cortex and cerebellum regions of each animal were dissected with the help of a stereotactic atlas (Paxinos, G., and Watson, C. (1986). Academic Press, New York, pp 230-259). A pool of each of these regions was formed and a homogenate of each region was prepared containing 1: 8 parts of tissue by 0.1 M phosphate buffer pH 7.0. The cell-free liquid was obtained by centrifugation at 4 degrees celcius at 10,000 g for 30 minutes. The homogenates were kept in an ice bath until the determination of the enzymatic activity, for which a Digital Spectro UV-VIS RS Spectrophotometer of Labo Med, Inc. As a main result it was obtained that the recombinant human Erythropoietin with low sialic acid content reduced by 13% (0-23%) the production of MDA + 4 HDA in the homogenates to the three regions studied in the brain of the rat. The decrease was dependent on the dose used. The highest levels of protection were detected in the hippocampus and cerebellum where considerable levels of EPO receptors have been detected. The response of the acidic and basic EPO was similar (Figure 9). Example 19:

The application of 90,000 Ul of recombinant human erythropoietin with low sialic acid content (according to formulation example 6) does not induce significant erythropoietic response.

To demonstrate that the recombinant human erythropoietin with low sialic acid content used in the trials has no significant erythropoietic activity "//? WVo" its effects were evaluated using a dose of 90 000 Ul on the erythropoiesis of the Mongolian Gerbils in a study comparative with 10 animals (5 administered and 5 controls), which were monitored (before administration, 7 days post administration and 14 days post administration) to perform a complete blood count in whole blood (10 μl_ of 10% EDTA / mL of blood) using a Micros ABX automatic counter for sample processing and the statistical package on Windows SPSS taking p = 0.05 as the confidence level for the processing of the variables.

The following results were obtained (Table 1).

Table 1. Hematological parameters obtained in the different groups.

The distribution of the populations for each hematological parameter by sampling with p = 0.05 and n = 10 showed a normal distribution, in the analysis of variance homogeneity it was observed that in a general way the variances had a homogeneous behavior, except for HB and HTC in sampling I, LEUC. In sampling Il and HCM and LEUC in sampling III. When comparing the variances of the hematological variables of the groups in the different samples, differences were observed in the HB, the ETO, the HTC and the CHCM of the sampling II, where differences between the control and the administered group are evidenced, when comparing the variances between the samples for each group, differences were observed in the CHCM of the control group (sampling I vs sampling II) and the treated group presented differences in HB and HTC (sampling Il vs. samples I and III) and the CHCM (sample Il vs sampling I ). For the evaluation of the differences of the aforementioned variables between the groups by sampling, sampling I was taken as reference sampling for the comparisons. Taking into account that there are some statistical differences p = 0.05 between the groups and between the samples, it was decided to perform a more comprehensive evaluation of the data obtained; for which one They grouped the values of sampling I, at which time the animals had not yet been administered n = 10, and the X + 2SD range of this population was calculated, graphing the behavior of the mean values for each variable (FIG. 10). The average values of the variables were also compared with those reported by other authors for this species, showing that in all groups and samples similar values were obtained to those reported (Table 2).

Although the statistical comparisons reflect that the variables: HB, ETO, and HTC increased significantly in sampling II, when analyzing this behavior according to the normal range X ± 2SD and the average values reported in the literature (Manual on care and use of experimental animals, Canadian Council of animal Protection. Volume 1, 2 ^nd edition. 1998), you may notice that this behavior has no biological significance, therefore they can be concluded that the recombinant human erythropoietin low Sialic acid does not induce a significant erythropoietic response in the species studied.

Table 2. Normal hematological values for Gerbils.

Example 20:

Marking of recombinant human erythropoietin with low sialic acid content with I ¹²⁵ .

To determine the passage of recombinant human erythropoietin with low sialic acid content (examples 3 and 5), to regions of the central nervous system when applied by the nasal route, the EPOrh was marked with sludge 125, according to the method described for the technique with YODO-GEN (Pierce. Rockford, Illinois 61105, USA). From this radioactive tide, formulations for nasal application were prepared. A total of 18 Wistar male rats of body weight between 180 and 20Og were used that were applied nasally with the referred formulation using the recombinant human Erythropoietin with low content of iodinated sialic acid with I ¹²⁵ . Each animal was applied between 5 and 100 microliths of the nasal formulation. At intervals of 5, 30 and 60 minutes the animals were sacrificed by decapitation under anesthesia. The brain was quickly removed (less than 70 seconds). The olfactory bulb and cerebellum areas were extracted. The radioactive count was performed on a Gamma counter. The following results were obtained: an application of recombinant human Erythropoietin with low sialic acid content reaches regions of the brain in at least 5 minutes and over time it gradually decreases from the frontal region to caudal. After 60 minutes of application, EPO remains in the olfactory bulb and cerebellum regions. These results indicate that approximately 1 to 5% of the nasally applied EPO reaches the brain. Between 80 and 85% of the EPO that reaches the brain was detected in the olfactory bulbs, while about 20% was detected in the cerebellum after 5 minutes of application. After 30 minutes these concentrations almost equalize and after 60 minutes the cerebellum has 30% and only 10% of the radioactivity of the olfactory bulb. The presence of the labeled molecule in regions of the cerebellum indicates the passage of the BHE, on the other hand, the detection of the molecule at the level of regions not related to the olfactory pathway (cerebellum) indicates that not only the olfactory pathway is compromised in the passage of molecules to the brain, but a more general and rapid mechanism is participating, which can be diffusion through the mucus and subsequent permeation through discontinuities that occur in regions of the olfactory epithelium, particularly in the area of the cribriform sheet , which allows the arrival of recombinant human erythropoietin with low content of sialic acid to regions far from the olfactory pathway. Figure 11 represents the passage through the nasal route of the recombinant human erythropoietin with low sialic acid content to olfactory bulb and cerebellum. The figure represents in each bar the average value of six animals. It shows how recombinant human erythropoietin with low sialic acid content reaches regions of the brain in at least 5 minutes and over time it gradually decreases from the frontal region at caudal. After 60 minutes of application, there is still EPO in the olfactory bulb and cerebellum regions. Between 80 and 85% of the EPO that reaches the brain was detected in the olfactory bulbs, while about 20% was detected in the cerebellum after 5 minutes of application. After 30 minutes these concentrations almost equalize and after 60 minutes the cerebellum has 30% and only 10% of the radioactivity of the olfactory bulb.

Example 21: Effect of basic EPOrh on subaranoid hemorrhage in rabbits

The test was carried out in male New Zealand rabbits with a weight between 3-4 Kg that were anesthetized with intramuscular ketamine 40 mg / kg, subsequently 5 ml of blood were extracted from the central artery of the ear that were percutaneously injected into the magna cistern , the animals were monitored for 72 h. The rabbits were divided into 3 experimental groups with 8 animals each: Group I: Non-injured animals Group II: Injured animals

Group III: Injured animals + basic EPOrh 20μg (example 5) The administrations of basic EPOrh were performed, 5 minutes after inducing the suranoid hemorrhage and repeated every 8 hours for 72 hours.

For the neurological evaluation, the motor behavior of the animals was followed according to Grasso G, 2002 during the trial period

The results are shown in Table I, observing a better behavior of the motor behavior in the control animals (without injury) and those treated with basic EPOrh than the animals injured without treatment.

The degree of neurological damage was classified as: 1: There are no indications of damage. 2: Minimal damage.

3: Moderate damage without abnormal movements. 4: Severe damage with abnormal movements

*: differs with respect to injured animals treated with basic EPOrh and non-injured animals for P <0.05

Example 22: Effect of the basic EPOrh for the treatment of dementia

The right carotid under anesthesia was linked to 36 glands in Mongolia and treated with 40 ul daily of EPOrh by nasal route (examples 10 and 11) for 7 days, beginning within 1 hour after surgery. Another 24 animals received similar processing and an equivalent amount of vehicle was administered. To 9 gérbiles the carotid was isolated without ligating it and constituted the control group.

At 8 days after the operation, 27 and 13 animals from the groups undergoing unilateral ischemia and treated with EPOrh and vehicle respectively had survived. There was no mortality for the control group. The animals were subjected to a habituation test one day before and one week after the unilateral ligation of the carotid. This test is based on the count of steepness in a cylindrical vessel for 9 minutes, divided into three thirds. The proportions of steepness in each third determine a line of best fit between the three points. Its slope was used to establish the comparisons between groups using the Mann and Whitney U test. Before-after comparisons were made by means of the Wilcoxon ranks comparison test of the surviving animals. The results were the following:

a - different from control; b - different from EPOrh; p <0.05.

The results show a smaller slope (closer to 0) in animals undergoing ischemia, one week after ischemia and in comparison with those treated with EPOrh and controls. A smaller slope demonstrates a persistence of the animal in exploring this environment in the last two thirds, showing a loss of habituation that can be characterized as a cognitive dysfunction induced by ischemia. The treatment with basic EPOrh prevents the onset of this dysfunction. The behavior of the animals revealed a deterioration induced by the ischemia that could be expressed in a loss of the ability to nest, typical in males of this species, in an increase in aggressiveness and in a loss of the grooming behavior during the time they remained in the circular cylinder. The results were compared using the chi-square independence test and were expressed in the table in percent:

a - different from control; b - different from EPOrh; p <0.05 The animals were sacrificed by infusion under anesthesia with 10% buffered neutral formalin. The brains were extracted and processed to obtain coronal sections of 8 μm stained with hematoxylin and eosin. The sections were examined under a light field microscope for the detection of histopathological signs. The comparisons were made by means of the chi-square independence test. The results of the table are expressed in percent:

a - different from control; b - different from EPOrh; p <0.05

It is notable that the treatment totally prevents edema and halves the incidence of picnosis, gliosis and neuronal loss.

The behavioral deterioration can be described as a reflection of the neuronal loss and other histopathological findings observed in the untreated group. The treatment with EPOrh prevents at least partially the behavioral deterioration induced by the decrease in cerebral blood flow in the gerbil of Mongolia. The results are relevant in the sense of suggesting a neuroprotective action of EPO rh capable of counteracting the functional deterioration of dementia of vascular origin in man.

Claims

1. Formulations from EPOrh with low sialic acid content characterized in that the finished form is administered by the nasal route.

2. Formulations from EPOrh with low sialic acid content according to claim 1 characterized in that the finished form are nasal drops.

3. Formulations from EPOrh with low sialic acid content according to claim 1 characterized in that the finished form is nasal spray.

4. Formulations from EPOrh with a low sialic acid content according to claim 1-3 characterized by the use of bioadhesive polymers such as hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC) and methylcellulose (MC).

5. Formulations from EPOrh with low content of sialic acid according to claim 1-3 characterized by the use of antimicrobial preservatives such as benzalkonium chloride, chlorobutanol, methylparaben and propylparaben.

6. Nasal formulations from EPOrh with low sialic acid content according to claims 1-3 characterized by the use of protein stabilizers such as L-tryptophan, L-leucine, L-arginine hydrochloride and / or L hydrochloride -histidine and / or its salts.

7. Formulations according to claim 1, to be used in the treatment of cerebrovascular, psychiatric, acute and chronic neurodegenerative diseases, hypoxia of the newborn, hypoxia due to cardiovascular causes, accidents, traumas and intoxication, dementia, loss of

The memory, decreased mental capacity, mental deterioration, neurological manifestations as a result of diseases, damage or alterations of the physiological system, psychiatric manifestations as a result of diseases, damage or alterations of the physiological system, neurological manifestations produced by peripheral diseases, psychiatric manifestations produced due to peripheral diseases, neurological manifestations produced by epileptic states.